The microbiota provides an important barrier to enteric infections and encodes microbe-associated molecular patterns as well as secondary metabolites, which can prime host immunity or attenuate pathogen fitness (Buffie and Pamer, 2013; Milshteyn et al., 2018). For example, polysaccharide A (PSA) from Bacteroides fragilis has been reported to activate Toll-like receptor two on FOXP3⁺ regulatory T cells, promote immunologic tolerance and enhance colonization of commensal bacteria (Round et al., 2011). Alternatively, butyrate production by Clostridial strains (clusters XIVa and IV) can attenuate inflammation by inducing peripheral regulatory T cells (Arpaia et al., 2013; Furusawa et al., 2013; Smith et al., 2013) and suppress reactive metabolites to prevent the expansion of enteric pathogens (Byndloss et al., 2017). In addition, commensal bacteria such as Clostridium scindens can generate secondary bile acids that mediate colonization resistance toward pathogens such as Clostridium difficile (Buffie et al., 2015). Other specific commensal strains such as Enterococcus faecalis (E. faecalis) contain plasmids such as pPD1 that produce bacteriocins, which can kill vancomycin-resistant enterococci and influence niche competition in the gut (Kommineni et al., 2015). While these studies have begun to reveal the functions of some specific commensal bacteria species, the mechanisms of action and specific protective factors for other commensal bacteria that are correlated with pathogen susceptibility in animals are still unknown.

E. faecium is a ubiquitous bacterium that has been recovered from the microbiota of many animals as well as humans (Van Tyne and Gilmore, 2014) and can even colonize the roundworm Caenorhabditis elegans (C. elegans) (Garsin et al., 2001). While pathogenic strains of E. faecium and E. faecalis are notable due to their acquisition of drug-resistance and nosocomial infections (Arias and Murray, 2012), commensal strains of E. faecium have been reported to protect animals from enteric pathogens and potentially improve host metabolism (Zheng et al., 2016). However, the specific protective factors and mechanisms of commensal E. faecium were unknown (Gilmore et al., 2014). To dissect the protective mechanisms of E. faecium, we utilized C. elegans as an animal model and discovered that diverse strains of E. faecium could protect worms against enteric pathogens (Rangan et al., 2016). E. faecium and its growth media effectively prevented Salmonella Typhimurium-induced pathogenesis in C. elegans. The protective activity of E. faecium growth media was protease sensitive, which led to the proteomic analysis of the supernatant and discovery of secreted antigen A (SagA) as one of the most abundant E. faecium secreted proteins. Remarkably, administration of recombinant SagA to C. elegans was sufficient to attenuate S. Typhimurium pathogenesis (Rangan et al., 2016). In addition, chromosomal insertion of sagA into non-protective E. faecalis (E. faecalis-sagA) enabled the expression and secretion of SagA, which also conferred protective activity against S. Typhimurium pathogenesis in C. elegans (Rangan et al., 2016). E. faecium and SagA did not affect S. Typhimurium colonization or replication in vivo, but rather required tol-1 signaling in C. elegans, suggesting the activation of host innate immunity was the key mechanism for enhanced tolerance against enteric pathogens (Rangan et al., 2016).

To evaluate whether E. faecium and SagA function through similar mechanisms in mammals, we evaluated germ-free and antibiotic-treated mouse models of infections. In these studies, E. faecium colonization, but not E. faecalis, improved intestinal barrier morphology, permeability, gene expression (Muc2, cryptdin2 and RegIIIγ) and prevented S. Typhimurium pathogenesis (Pedicord et al., 2016). E. faecium protection against S. Typhimurium did not involve adaptive immunity mechanisms, but required innate immune signaling factors such as MyD88 (Toll-like receptor adaptor) and NOD2 as well as RegIIIγ, a key effector of intestinal barrier function (Pedicord et al., 2016). E. faecalis-sagA also increased the expression of intestinal epithelial barrier function genes (Muc2, cryptdin2 and RegIIIγ) and enhanced bacterial segregation from intestinal epithelial cells in vivo similar to E. faecium. Moreover, SagA can be expressed in probiotic bacteria such as Lactobacillus plantarum and enhanced its protective activity against S. Typhimurium as well as C. difficile (Pedicord et al., 2016; Rangan et al., 2016). These studies demonstrated that E. faecium and SagA-expressing bacteria were sufficient to enhance intestinal barrier function and prevent infection by diverse Gram-negative and Gram-positive enteric pathogens, but the biochemical mechanism(s) by which innate immunity was activated was unknown.

Here, we demonstrated that E. faecium peptidoglycan contains smaller non-crosslinked muropeptides compared to non-protective strains of E. faecalis, which more effectively activated the intracellular peptidoglycan pattern recognition receptor NOD2. In addition, we determined the X-ray structure of the E. faecium SagA-NlpC/p60 catalytic domain and showed that it encodes L-Lys-type endopeptidase activity. The SagA-NlpC/p60 domain selectively cleaved purified crosslinked peptidoglycan fragments into smaller muropeptides, which were more active towards NOD2 in mammalian cells. Furthermore, we demonstrated that the secretion and peptidoglycan hydrolase activity of SagA was required to enhance probiotic bacteria activity against C. difficile infection in vivo. Our structural, biochemical, cellular and in vivo studies revealed how E. faecium and its secreted peptidoglycan hydrolase SagA activate innate immunity in mammals and provide a mechanistic basis by which commensal bacteria modulation of peptidoglycan composition confers host protection against enteric infections.

Since E. faecium protection against S. Typhimurium was abrogated in Nod2-/- mice in vivo and SagA contains predicted peptidoglycan hydrolase (Pedicord et al., 2016), we focused on whether E. faecium and SagA exhibit unique peptidoglycan composition and activity. As sagA was shown to be essential for E. faecium growth (Teng et al., 2003), we focused on the characterization of E. faecium (Com15), E. faecalis (OG1RF), and E. faecalis-sagA (Rangan et al., 2016), in which the sagA gene was chromosomally inserted downstream of mreD and expressed under the endogenous sagA promoter. To directly compare SagA protein expression levels in these strains, we generated polyclonal sera against full length recombinant SagA (Figure 1—figure supplement 1) and confirmed that there were comparable levels of SagA expression in the cell pellets and secretion in the supernatants of E. faecium and E. faecalis-sagA, which was absent in E. faecalis (Figure 1a). Analysis of the bacterial growth rates showed that E. faecium divided most rapidly and reached optical density values of up to OD_600nm = 2.5 after 4 hr (Figure 1b). During lag phase, there was no marked difference in E. faecium, E. faecalis, and E. faecalis-sagA. However, E. faecalis-sagA exhibited a decreased growth rate compared to E. faecalis after 3 hr, resulting in slower exponential growth (Figure 1b). Analysis of these bacterial strains by transmission electron microscopy showed that E. faecium exhibited unique cell wall morphology compared to E. faecalis (Figure 1c). Interestingly, E. faecalis-sagA exhibited a significant increase in bacterial cell width as well as the presence of minor deformations to the integrity of the cell wall structure (Figure 1c,d), suggesting SagA expression may regulate cell wall composition and integrity in Enterococcus.

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To evaluate whether SagA expression affects bacterial cell wall composition, we isolated peptidoglycan from log-phase E. faecium, E. faecalis, and E. faecalis-sagA and analyzed the mutanolysin-digested peptidoglycan fragments by LC-MS/MS as previously described (Kühner et al., 2014). The major monomeric and crosslinked muropeptides we isolated from E. faecium and E. faecalis were similar to previous studies (Billot-Klein et al., 1996; Emirian et al., 2009; Patti et al., 2008), which showed that E. faecium generally contains non crosslinked muropeptides, including GlcNAc-MurNAc-L-Ala-D-isoGln (GlcNAc-MDP) compared to E. faecalis (Figure 2a). Interestingly, the analysis of peptidoglycan from E. faecalis-sagA revealed a decrease in muropeptide fragments (peak 2, 7, 9, 13, and 17) compared with E. faecalis (Figure 2a, Figure 2—figure supplement 1a,b and Supplementary files 1 and 2). To evaluate whether these changes in bacterial growth, cell morphology and peptidoglycan composition were associated with SagA expression and not other mutations generated during chromosomal sagA insertion, we performed whole genome sequencing on both E. faecalis and E. faecalis-sagA. This analysis confirmed the insertion of sagA downstream of mreD, as originally designed, and also revealed a total of 39 genes with synonymous and non-synonymous mutations ranging from 25 to 100 percent coverage (Supplementary file 3). However, none of the non-synonymous mutations greater than 50 percent coverage were in genes directly associated with bacterial cell wall synthesis or remodeling, suggesting SagA expression affected growth, morphology and peptidoglycan composition of E. faecalis-sagA.

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We then tested whether E. faecium, E. faecalis and E. faecalis-sagA could activate peptidoglycan pattern recognition receptors. In mammals, NOD1 and NOD2 are the predominant sensors of peptidoglycan fragments and are reported to recognize gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP) and muramyl-dipeptide (MDP), respectively (Caruso et al., 2014; Philpott et al., 2014). Peptidoglycan activation of intracellular NOD1 and NOD2 triggers the expression of NF-κB-responsive genes associated with host immunity and inflammation (Caruso et al., 2014; Philpott et al., 2014). To evaluate the activation of these receptors, increasing amounts of E. faecium, E. faecalis and E. faecalis-sagA were added to NOD1 or NOD2-transfected HEK293T cells co-expressing NF-κB luciferase reporters. Compared to synthetic ligands iE-DAP and MDP, E. faecium with a multiplicity of infection (MOI = 1) activated NOD2, but not NOD1 (Figure 2b). Higher MOI of E. faecium resulted in the detachment of transfected HEK293T cells (data not shown). In contrast, E. faecalis and E. faecalis-sagA required 10³-fold more bacteria (MOI = 10³) to activate NOD2, but not NOD1 (Figure 2b). The selective activation of NOD2 is consistent with its agonist specificity for Lys-type muropeptides, which were found in Enterococci peptidoglycan (Billot-Klein et al., 1996; Emirian et al., 2009; Patti et al., 2008). While the chromosomal expression of SagA altered the peptidoglycan composition of E. faecalis (Figure 2a), these differences did not appear to be sufficient to enhance direct bacterial activation of NOD2 in HEK293T cells. The differences in NOD2 activation by E. faecium compared to E. faecalis and E. faecalis-sagA may be due to enhanced bacterial adhesion and/or internalization since more E. faecium was recovered from gentamicin protection assays (Figure 2c). These results showed E. faecium contained smaller muramyl peptides such as GlcNAc-MDP and may directly interact with mammalian cells to activate NOD2.

To further dissect the protective activity of E. faecium and SagA, we focused on the activity of purified SagA and soluble peptidoglycan fragments. SagA encodes an N-terminal signal sequence, predicted coiled-coil domain, Ser/Thr-rich linker region and C-terminal NlpC/p60-family peptidase domain (Figure 3a). Bioinformatic analysis suggested that SagA is unique to E. faecium (Figure 3—figure supplement 1), with no clear homologues with greater than 25% protein sequence identity in most sequenced strains of E. faecalis (Neumann et al., 2019; Palmer et al., 2010). Nonetheless, the SagA from E. faecium Com15 does share significant protein sequence similarity to other NlpC/p60 orthologs in other E. faecium strains (Figure 3—figure supplement 2). Indeed, we demonstrated that the NlpC/p60-family peptidase domain and conserved active site residues were required for SagA-mediated protection in C. elegans (Rangan et al., 2016). However, the precise peptidoglycan substrates of SagA were unknown.

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To directly evaluate the peptidoglycan hydrolase activity of SagA, we expressed and purified full-length His-tagged SagA and truncated C-terminal His-tagged constructs along with C443A mutants (Figure 3—figure supplement 3a,b). These recombinant SagA constructs did not cleave purified intact E. faecium peptidoglycan, but incubation of the SagA-NlpC/p60 domain with mutanolysin-digested peptidoglycan yielded small muropeptides by in-gel fluorescence profiling (Figure 3—figure supplement 3c). Intriguingly, the SagA-NlpC/p60 domain only cleaved mutanolysin-digested peptidoglycan from E. faecium and E. faecalis, but not E. coli (Figure 3—figure supplement 3d), which suggested that it may be specific for L-Lys-type peptidoglycan found in Enterococcus versus mDAP-type in E. coli. LC-MS analysis of the full-length SagA and SagA-NlpC/p60 reaction products revealed the generation of several new peptidoglycan fragments as enzymatic products such as tripeptide (peak a), GlcNAc-MDP (peak b), GlcNAc-MurNAc-tri-di (peak c), GlcNAc-MurNAc-tetra-tri (peak d) and larger fragments (peak e) (Figure 3—figure supplement 3e and Supplementary file 4), which were absent when the full-length SagA-C443A and the SagA-NlpC/p60-C443A active site mutants were added. Full-length SagA expressed and purified from E. coli at similar protein concentrations was less active with mutanolysin-digested peptidoglycan than truncated SagA-NlpC/p60 domain (Figure 3—figure supplement 3e), suggesting the N-terminus may inhibit or modulate the activity of NlpC/p60 domain, akin to the coiled-coil domain of PcsB from Streptococcus pneumonia (Bartual et al., 2014).

To understand the molecular mechanism of E. facium SagA-NlpC/p60, we determined crystal structure of the E. faecium SagA-NlpC/p60 domain. Gel-filtration analysis revealed that SagA-NlpC/p60 is monomeric (Figure 3b) and amenable to crystallization (Supplementary Results). The structure of the SagA-NlpC/p60 domain was solved to 2.4 Å resolution by using X-ray crystallography. The data were indexed in space group P432 with one molecule per asymmetric unit (asu) and the initial phases was estimated by molecular replacement method by using Staphylococcus aureus CwlT (PDB 4FDY) as a search model. The electron density was well defined for the majority of the protein residues. Flexible segments of the first 26 residues, the His₆-tag, and some side chains were disordered and not included in the final model (Figure 3c,d PDB ID: 6B8C, Supplementary file 5). The SagA-NlpC/p60 domain contained the typical features of the cysteine peptidase fold (Anantharaman and Aravind, 2003), a central six-stranded, antiparallel β-sheet flanked by three surrounding α-helices (Figure 3c). The overall dimensions of the structure were about 32.5 × 30×23 Å (Figure 3d) and shared significant structural homology to other NlpC/p60 hydrolase domains. The root-mean-square deviations between superimposed Cα atoms was a range of 1.6 ~ 2.0 Å when comparing with NlpC/p60 hydrolase domains of other structures (Supplementary file 6 and Figure 3—figure supplement 4). The conserved catalytic triad (C443, H494 and H506) of the SagA-NlpC/p60 domain was located in a putative substrate-binding groove between the subdomains in the α2 and β3–β4 (Figure 3c,d), where electron density for these key amino acid residues were well observed (Figure 3e). An electrostatic potential analysis of the SagA-NlpC/p60 domain showed that the putative substrate-binding groove was mostly composed of negatively charged amino acid residues (Figure 3f). Homology-based structural search using ConSurf server suggested that the residues with the highest percentage of conservation were in the active site in the putative substrate binding pocket. Structural alignments with other homologs also showed similar conformations of the catalytic triad in the active site to that of the YkfC from Bacillus cereus (Xu et al., 2010), NpPCP from Nostoc punctiforme (Xu et al., 2009), AvPCP from Anabaena variabilis (Xu et al., 2009), CwlT from Staphylococcus aureus (Xu et al., 2014a), Spr from Escherichia coli (Aramini et al., 2008), RipA from Mycobacterium tuberculosis (Ruggiero et al., 2010), and LysM from Thermus thermophilus (Wong et al., 2015) active sites (Figure 3g).

To explore how the SagA-NlpC/p60 domain may interact with peptidoglycan substrates, we compared our structure to other NlpC/p60 hydrolases and performed potential substrate docking studies. While no structure of a NlpC/p60 hydrolase bound to peptidoglycan substrates has been determined, previous docking studies of CwlT suggested electropositive regions of these enzymes may be involved in substrate binding (Xu et al., 2014a). Indeed, the overlay of our SagA-NlpC/p60 structure with CwlT suggests potential conserved regions of substrate binding (Figure 4a and Figure 3—figure supplement 4). We then performed docking studies of solved structure with peptidoglycan fragments using Grid-based Ligand Docking with Energetics (GLIDE) module of the Schrödinger (Friesner et al., 2006). Amongst the peptidoglycan fragments we explored, GlcNAc-MurNAc-L-Ala-D-isoGln-L-Lys-D-Ala (Figure 4b,c) and the corresponding tetrapeptide alone (Figure 4—figure supplement 1) afforded a set of ligand-bound poses in the putative substrate-binding groove with high-scoring calculated binding free energies from 1 to 50 (Supplementary file 7). Manual inspection of the top-posed GlcNAc-MurNAc-tetrapeptide docked into the SagA-NlpC/p60 domain showed the predicted active site cysteine (C443) and histidine residues (H494 and H506) were well positioned near the potential site of peptide bond cleavage (Figure 4c, pose one from Supplementary file 7) and suggested peptidoglycan fragments may extend across the putative substrate-binding groove to interact with electropositive amino acid residues on the potential glycan and peptide binding sites at the surface (Figure 3f). Interestingly, the SagA-NlpC/p60 ligand-docked models also suggested that two tryptophans (W433 and W462) may serve as clamps for binding potential peptidoglycan substrates (Figure 4b,c). Indeed, single and double Ala mutants of W433 and W462 significantly abrogated hydrolytic activity of SagA-NlpC/p60 domain (Figure 4—figure supplement 2). These results demonstrate C443, W433 and W462 are crucial for the SagA-NlpC/p60 peptidoglycan hydrolase activity and encompass the enzyme active site as well as putative substrate-binding groove.

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To identify the precise substrate of SagA-NlpC/p60, we further isolated muropeptides (disaccharide-tripeptide, disaccharide-tetrapeptide, disaccharide-pentapeptide, and crosslinked disaccharide-tripeptide-disaccharide-tetrapeptide) from E. faecium peptidoglycan. Disaccharide-tripeptide, disaccharide-tetrapeptide and disaccharide-pentapeptide were not cleaved or only showed modest production of GlcNAc-MDP (Figure 4—figure supplement 3a,b,c). However, incubation of SagA-NlpC/p60 with disaccharide-tripeptide crosslinked to disaccharide-tetrapeptide readily generated GlcNAc-MDP and the corresponding GlcNAc-MurNAc-L-Ala-D-isoGln-L-Lys-crosslinked-D-Ala-L-Lys heptapeptide product (GlcNAc-M7P) (Figure 4d) and Figure 4—figure supplement 3d) with a specific activity of approximately 67 ± 7 nmol/min/mg at 37°C (Figure 4e), which did not occur with the NlpC/p60-C443A mutant (Figure 4—figure supplement 3d). The identity of GlcNAc-MDP was further confirmed by LC-MS/MS analysis and its fragmentation into MDP (Figure 4—figure supplement 4b,c). These results demonstrated that SagA-NlpC/p60 encodes cysteine endopeptidase activity that prefers crosslinked peptidoglycan fragments and cleaves the peptide bond between D-iGln and L-Lys (Figure 4f).

We next evaluated the activity of the crosslinked muropeptide substrate and SagA-NlpC/p60-generated muropeptides, GlcNAc-MDP and GlcNAc-M7P, on NOD1 and NOD2 activation in mammalian cells. To control for endogenous activation, non-transfected parental HEK-Blue-Null2 cells were also used as a negative control (Figure 5a). None of SagA-generated ligands activated NOD1 (Figure 5a). We chemically synthesized GlcNAc-MDP (Figure 5—figure supplement 1) and directly compared its activity to SagA-generated GlcNAc-MDP and commercial MDP as a positive control. Using NOD1 and NOD2 HEK-Blue cells that exhibited better dose-response to ligands, we found that SagA-generated and synthetic GlcNAc-MDP activated NOD2 in a dose-dependent manner comparable to commercial MDP standard (Figure 5b). SagA-NlpC/p60-generated muropeptides, GlcNAc-MDP most effectively activated NOD2, and GlcNAc-M7P less effectively and both ligands were more active than the crosslinked peptidoglycan fragment (Figure 5b). These results demonstrated that SagA-generated muropeptides can directly activate NOD2 in mammalian cells, consistent with previous studies of MDP derivatives (Dagil et al., 2016; Davis et al., 2011; Fujimoto et al., 2009; Girardin et al., 2003b; Wang et al., 2013), which suggests that GlcNAc-MDP derivatives are further processed by hydrolytic enzymes to generate MDP in mammalian cells. Importantly, these results indicated non-crosslinked muropeptides more effectively activate NOD2 in mammalian cells and provides a biochemical mechanism by which E. faecium and secreted SagA may enhance host immunity toward enteric pathogens.

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To determine if SagA cleavage of peptidoglycan is important in vivo, we evaluated the protective activity of bacteria expressing wild-type or SagA mutants in mouse model of C. difficile infection. Since sagA is essential in E. faecium (Teng et al., 2003) and oral administration of recombinant SagA was not effective (data not shown), we focused on heterologous expression of SagA variants in E. faecalis and the probiotic bacteria L. plantarum (Lpl) (WCSF1). While chromosomal insertion of sagA into E. faecalis was functional (Rangan et al., 2016), insertion of a SagA active site mutant yielded low or undetectable levels of protein expression in cell lysate or supernatant (Figure 5—figure supplement 2). Overexpression of SagA variants on plasmids in E. faecalis were either toxic or did not yield adequate levels of protein expression (data not shown). However, plasmid-based expression of SagA-His₆ variants in L. plantarum yielded comparable levels of wild-type SagA, signal sequence mutant (ΔSS) as well as active site mutant (C443A) in cell lysates, with reduced levels of ΔSS mutant in the supernatant (Figure 5c). Heterologous expression of these SagA variants did not significantly alter the L. plantarum peptidoglycan composition (amidated mDAP-type) (Goffin et al., 2005) (Figure 5—figure supplement 3). However, colonization of antibiotic (Abx)-treated mice with these L. plantarum strains showed that wild-type SagA protected mice from C. difficile pathogenesis, while the SagA signal sequence (ΔSS) as well as active site (C443A) mutant were not protective and comparable to L. plantarum-vector and PBS controls (Figure 5d and Figure 5—figure supplement 4). These results demonstrate that SagA secretion and peptidoglycan endopeptidase activity are required for enhancing the activity of probiotic bacteria against enteric pathogens in vivo.

The microbiota and specific strains of commensal and probiotic bacteria have been suggested to activate peptidoglycan pattern recognition receptors in vivo (Caruso et al., 2014; Philpott et al., 2014). Moreover, deletion or loss-of-function mutations of NOD2 in particular are associated with gut microbiota dysbiosis, invasion of pathobionts and inflammatory bowel disease (IBD) (Caruso et al., 2014; Philpott et al., 2014), which suggests that NOD2 is important for sensing peptidoglycan in the gut and priming innate immune pathways to maintain intestinal barrier function and preventing microbiota- and pathogen-induced inflammation (Al Nabhani et al., 2017; Caruso et al., 2014; Philpott et al., 2014). Indeed, our previous studies showed that NOD2 was required for E. faecium-mediated protection against enteric pathogen infection (Pedicord et al., 2016). However, the mechanism by which E. faecium and its secreted peptidoglycan hydrolase SagA engaged NOD2 was unknown.

To dissect the biochemical mechanism(s) by which E. faecium and SagA enhances intestinal barrier function and protects against enteric pathogens, we evaluated E. faecium peptidoglycan composition, SagA structure, their associated muropeptide products, activation of peptidoglycan pattern recognition receptors in mammalian cells. The comparative analysis of the peptidoglycan composition and NOD2 activity between bacteria suggested that E. faecium more effectively activated NOD2 through non-crosslinked muropeptides such as GlcNAc-MDP that was not present in E. faecalis and L. plantarum, even with SagA expression (Figure 2 and Figure 5—figure supplement 3). The results suggested E. faecium may be intrinsically more capable of interacting with mammalian cells and/or directly activate NOD2 even though both Enterococci species have Lys-type peptidoglycan. Nonetheless, SagA expression still enhances the protective activity of E. faecalis and L. plantarum against enteric pathogens in vivo (Pedicord et al., 2016; Rangan et al., 2016), suggesting secreted SagA may also prime or activate immune pathways.

To characterize the activity of secreted SagA, we expressed and purified recombinant SagA and investigated on the structure and enzymatic activity of C-terminal NlpC/p60 hydrolase domain. Although full-length SagA purified from E. coli was active, we focused on the C-terminal NlpC/p60 hydrolase domain since it was significantly more active in vitro. X-ray crystallography showed that the SagA-NlpC/p60 domain was indeed similar to other bacterial hydrolases and shared similar active site residues (Figure 3). Analysis of SagA-NlpC/p60 domain with purified peptidoglycan fragments demonstrated that it showed endopeptidase activity and preferentially cleaved L-Lys-type crosslinked peptidoglycan fragments (Figure 4), which required the conserved active site Cys443. In addition, W433 and W462 were also essential for the endopeptidase activity in vitro and may be involved in stabilization of the peptidoglycan substrate binding based on our SagA-NlpC/p60 structure and docking studies. These tryptophan residues or other hydrophobic aromatic amino acids are highly conserved amongst other SagA orthologs in Enterococci as well as other hydrolases in other bacteria (Figure 3—figure supplements 1 and 2). Beyond these highly conserved amino acid residues in the SagA-NlpC/p60 active site, the molecular basis for Lys- or DAP-type peptidoglycan specificity is still unclear from ours and other existing NlpC/p60 hydrolases structures (Xu et al., 2009; Xu et al., 2010; Xu et al., 2014b; Xu et al., 2015). Additional studies of these SagA-NlpC/p60 mutants with crosslinked muropeptides are therefore in progress to fully understand their roles in catalysis and substrate recognition. Further studies of full-length SagA are also needed to investigate its structure and activity as well as potential auto-inhibition by the N-terminal coil-coil domain observed in other NlpC/p60 hydrolases (Bartual et al., 2014). For example, the X-ray structure of full-length Streptococcus pneumoniae, PcsB, another class of peptidoglycan hydrolase that contains an N-terminal coil-coil domain, suggest this domain facilitate its dimerization and regulate activity as well as bacterial cell division (Bartual et al., 2014). The function of SagA and its orthologs in Enterococci peptidoglycan turnover and cell division remains to be determined, since sagA is essential in E. faecium (Teng et al., 2003) and requires new approaches for conditional inactivation for further studies.

Notably, these studies demonstrated that SagA-NlpC/p60 generated non-crosslinked muropeptides such as GlcNAc-MDP, which were also found in E. faecium peptidoglycan and activated NOD2 in mammalian cells more effectively than larger and crosslinked muropeptides (Figure 5). In addition to these in vitro biochemical studies and ex vivo activation of NOD2 in mammalian cells, we demonstrate that the secretion and endopeptidase activity of SagA is required for enhancing the protective activity of L. plantarum against C. difficile infection in vivo (Figure 5d). Collectively, our results suggest that E. faecium and its secreted peptidoglycan hydrolase SagA generates small muropeptides that activate NOD2, which triggers downstream signaling pathways to enhance intestinal barrier function and host immunity (Figure 6). Although MDP were not active in C. elegans, which does not have a clear NOD2 ortholog, MurNAc and MurNAc-L-Ala were sufficient to attenuate bacterial pathogenesis and required the Tol-1 signaling (Rangan et al., 2016), suggesting the recognition of small bacteria-specific glycans may also be conserved in nematodes but utilize different receptors akin to insects (Dziarski and Gupta, 2006). In mammals, smaller muropeptide fragments have been reported to enhance NOD2 activation ex vivo (Dagil et al., 2016; Davis et al., 2011; Fujimoto et al., 2009; Girardin et al., 2003a; Wang et al., 2013) and function as more effective adjuvants for vaccination (Hancock et al., 2012; Ogawa et al., 2011; Rubino et al., 2013) or enhance monocyte activity in vivo (Coulombe et al., 2012; Namba et al., 1996). Since NOD2 is important for modulating immune signaling in many areas of host physiology and disease (Caruso et al., 2014; Keestra-Gounder and Tsolis, 2017; Philpott et al., 2014), further analysis of Enterococci and SagA orthologs will be important for understanding specific microbiota-host interactions and may afford new opportunities for therapeutic development.

Figure 6

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All data generated or analyzed during this study are included in the manuscript and supporting files. Diffraction data have been deposited in PDB under the accession code 6B8C.

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Author details

1. Byungchul Kim

   Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0207-7357

2. Yen-Chih Wang

   Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

3. Charles W Hespen

   Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5466-0280

4. Juliel Espinosa

   Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2104-5331

5. Jeanne Salje

   Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

6. Kavita J Rangan

   Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

7. Deena A Oren

   Structural Biology Resource Center, The Rockefeller University, New York, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

8. Jin Young Kang

   Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States

   Contribution

   Formal analysis, Validation, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

9. Virginia A Pedicord

   1. Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, United States
   2. Cambridge Institute of Therapeutic Immunology & Infectious Disease, University of Cambridge, Cambridge, United Kingdom

   Present address

   Cambridge Institute of Therapeutic Immunology & Infectious Disease (CITIID), University of Cambridge, Cambridge, United Kingdom

   Contribution

   Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

10. Howard C Hang

    Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, United States

    Contribution

    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

    For correspondence

    hhang@rockefeller.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-4053-5547

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